RNA-seq after DDX41 knockdown in U2OS cells
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ABSTRACT:
PROVIDER: PRJNA706394 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: Investigating transcriptome changes after depletion of DDX41. Methods: mRNA profiles after DDX41 knockdown compared to control knockdown were generated in 5 biological replicates. Samples were mapped using STAR (v2.7) against hg38 with the Gencode annotation. Reads per gene were counted using featureCounts (v.1.6). The differential expression analysis was performed using Bioconductor (v2.46)/DESeq2 (v1.26). Genes were deemed sign. diff. regulated with an FDR below 1% Results: We were able to map 32,705 transcripts. Reactome pathway analysis revealed members of the NOTCH and TGFß family to be significantly upregulated when comparing a control knockdown to DDX41 knockdown. In addition, genes in chromatin organization were upregulated.
2021-05-04 | GSE168171 | GEO
Project description:Purpose: Investigation of genome-wide changes in R-loop levels after knockdown of DDX41 U2OS cells in comparison to a control knockdown. Method: Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2). 5*105 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 50 µl Wash Buffer containing 0.05% Digitonin. Either pA-MNase or RHΔ-MNase were added to the cells overnight at 4°C on a rotating wheel. After three washes with Wash Buffer containing 0.05% Digitonin, samples resuspended in 100 µl Dig-Wash-Buffer were equilibrated on ice. Activity of the MNase was triggered by adding 2mM CaCl2 to the samples for 30 minutes. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, 20 pg/ml spike-in Drosophila DNA (Active Motif)) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We were able to map R-loop dynamics genome-wide in U2OS cells in biological triplicates. MapR signal was significantly increased around the transcription start site in the absence of DDX41 and decreased after treatment with ActinomycinD.
2021-05-04 | GSE168170 | GEO
Project description:Purpose: Investigation of DDX41 chromatin binding sites in U2OS cells Method: Expression of DDX41-GFP was induced for 48 hours before crosslinking using 1µg/ml docycycline. Control cells were not induced with doxycline. Cells were mildly crosslinked using 1% FA for 2 min at RT. Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2).1*10^6 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 1 ml Wash Buffer. Cells were permeabilized with 0.05% digitonin in Wash buffer for 4 min at RT. 1 µg Nanobody-GFP-MNase (in-house protein production, PMID:25362362) binding was performed at 4°C for 30 min in 0.05% digitonin-Wash buffer. After 2x washing, the MNase was activated by adding 3mM CaCl2 on ice for 30 min. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, )) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We succesfully mapped DDX41 binding sites on cromatin in U2OS cells. DDX41 preferentially binds in the promoter region of genes.
2021-11-17 | GSE183083 | GEO
Project description:Frac-Seq Analysis of ZFC3H1 Knockdown U2OS Cells
| PRJNA735123 | ENA
Project description:We report the first genome-wide mapping of fragility hotspots after siRNA-mediated knockdown of the DEAD-box helicase DDX41 in HCT116 cells using sBLISS. DDX41 defficient cells display elevated levels of genomic instability. sBLISS analysis revealed that hotspots for genomic instability in the absence of DDX41 are located around the transcription start site of transcribed genes.
2021-11-17 | GSE183147 | GEO
Project description:Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-Box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases lead to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cryopreserved diagnostic bone marrow or peripheral blood samples.
2015-04-01 | E-GEOD-66668 | biostudies-arrayexpress
Project description:To gain insight into the function of both OCIAD paralogs, we used untargeted quantitative mass spectrometry to compare the whole-cell proteomes of control U2OS cells, U2OS cells with individual or double OCIAD1/OCIAD2 knockdown, and OCIAD1 knockdown cells in which OCIAD1 expression was reintroduced by lentiviral delivery.
2021-05-14 | PXD025573 | Pride
Project description:MapR in U2OS and HCT116 cells and RNA-seq in U2OS
| PRJNA706388 | ENA
Project description:Homologous recombination-mediated DNA repair deficiency (HRD) predisposes to cancer development, but also provides therapeutic opportunities. Here, we identified an HRD gene signature that robustly predicted HRD status. Unexpectedly, concurrent loss of PTEN in BRCA1-deficient cells might extensively rewire the HR repair network and confer resistance to PARP inhibitor, partially through over-expression of TTK. We used the HRD gene signature as a drug discovery tool and found several PARP-inhibitor-synergizing agents through the connectivity map. Thus gene expression profiling can be used to define the functional status of the HR repair network providing prognostic and therapeutic information. Various shRNAs that target genes involved in homologous recombination (HR) were transfected in MCF-10A non-transformed breast cells lines. Stable HR gene knockdown MCF-10A cells were seeded 200000 at 10 cm plate. Cells were harvested after 48 hours culturing and used for gene expression profiling. The shRNA that target CHK1 gene was transfected in human osteosarcoma U2OS cell line by lentiviral particles and selected stable CHK1 knockdown U2OS cells. Scrambled control shRNA-transfected U2OS cells were applying as control. Both stable CHK1 knockdown and control U2OS cells were seeded with 2 x 10^5 cells at 10 cm culture plate. Cells were cultured in McCOY 5A medium with 10% FBS and harvested after 48 hours culturing. mRNA was extracted from collected cells and performing gene expression profiling. Three biological replicates were applied.
2014-01-24 | E-GEOD-54267 | biostudies-arrayexpress
Project description:DDX41, a member of the DEXDc family of helicases and an innate immune protein, senses cytosolic DNA and bacterial secondary messengers and initiates signaling via the adaptor STING to induce type 1 interferon (IFN) response in dendritic cells. However, DDX41 function in tumor progression is poorly understood. Here we found that the DDX41 inhibited proliferation and promoted apoptosis, reported the whole transcriptome profiling in HeLa cells. RNA-seq analyses revealed that the overexpression of DDX41 resulted in 959 genes being differentially expressed (504 up-regulated and 455 down-regulated) compared to the control in HeLa cells. Interestingly, functional clustering pathway enrichment analysis of transcription identified antigen processing and presentation pathways were significantly activated in DDX41 overexpression samples, but alternative splicing enriched in the epidermal growth factor receptor signaling pathway and fibroblast growth factor receptor signaling pathway. The five antigen processing and processing genes, which could regulate cross-presentation of antigens and protective antitumor immune responses, were significantly upregulated, suggesting DDX41 is the key player in tumor immunity. In conclusion, our RNA-seq data identified molecules and pathways involved in the mechanisms of DDX41 that adds to the understanding of critical DDX41 tumorigenesis functions.
2021-02-10 | GSE122986 | GEO